Date of Award

Winter 1994

Project Type

Dissertation

Program or Major

Chemistry

Degree Name

Doctor of Philosophy

First Advisor

N Dennis Chasteen

Abstract

The functional roles of ferritin H and L subunits in ferrous iron oxidation and ferric iron hydrolytic polymerization were studied by combination of electrode oximetry and pH stat utilizing horse spleen and recombinant human H and L ferritins. Previous work has investigated ferrous ion oxidation kinetics, but no information on ferric core formation and its relationship to ferrous ion oxidation had been obtained. The kinetic pattern of iron oxidation/hydrolysis in H-chain homopolymer ferritin showed that the rate of iron hydrolysis is regulated by the rate of iron oxidation, and that both follow the iron ferroxidation pathway. In the early stage of the reaction with less than 10 Fe(II)/protein, it was observed that the stoichiometric ratio of proton release to ferrous iron consumption approaches zero, the ferrous iron to dioxygen ratio equals two and the kinetic ratio of proton release to dioxygen consumption approaches one. These results enable one to write a detailed mechanism for iron ferroxidation occurring in ferritins containing the H-subunit. The L-chain homopolymer ferritin was also shown to facilitate iron oxidation, but iron deposition occurs by a mineral core surface mechanism as evidenced by the kinetic ratio of eight protons released per dioxygen consumed. These results suggest that the H-subunit serves as a ferrous iron oxidation source and the L-subunit acts as a ferric iron sink during iron incorporation into native heteropolymer ferritins.

Two types of ferritin radicals were observed by epr spectroscopy. One radical is a tyrosyl radical formed in the H-subunit only and is possibly generated from a superoxide intermediate produced during the one electron oxidation of iron(II). This radical gives a characteristic tyrosyl radical doublet with a g-factor of 2.0066 and a hyperfine splitting of 18 G. This radical is centered on a Tyr-34 as shown by measurements with site directed mutants. The other type of ferritin radical produced is a secondary radical derived from hydroxyl radical attack on ferritin. This radical is possibly formed on an iron binding site of the L-subunit. Neither of these ferritin radicals play an important role in iron ferroxidation. However, confinement of radical production to the inside of the ferritin shell could be a protective mechanism against iron toxicity.

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